Proteolytic processing of peptides in the lumen of the endoplasmic reticulum for antigen presentation by major histocompatibility class I

We have tested the hypothesis that MHC class I molecules are actively invol ved as protease in the production of natural MHC class I ligands. First, th e structure of a class I molecule was analyzed for homology with catalytic sites of known proteases. While several clusters of amino acids in the rest riction element resembled protease active sites, structural discrepancies a nd the influence of nearby residues suggest that these sites are unlikely t o have protease activity. Second, we have tested the presentation of viral cytotoxic T cell determinants with affinity for the same restriction elemen t (H-2K(d) or K-k), when targeted as tandem peptides into the endoplasmic r eticulum. Peptide transporter-defective cells were used to exclude cleavage of the tandem peptides by cytosolic proteases. Cleavage by signal peptidas e of the tandem peptides was ascertained. The C-terminal peptides in the ta ndem arrays were almost exclusively presented, suggesting that an aminopept idase in the endoplasmic reticulum degraded the N-terminally positioned pep tides. This result is inconsistent with an MHC class I-catalyzed cleavage f ollowing binding of longer peptides in the cleft of the restriction element s. Finally, we conclusively show that an aminopeptidase in the endoplasmic reticulum is also involved in antigen presentation in cells with a function al peptide transporter.